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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new xanthone derivative from twigs of Garcinia nobilis ab
c
d
Hugues Fouotsa , Simplice J.N. Tatsimo , Beate Neumann , b
a
Carmela Michalek , Celine Djama Mbazoa , Augustin Ephrem a
b
bc
Nkengfack , Norbert Sewald & Alain Meli Lannang a
Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon b
Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany c
Department of Chemistry, Higher Teachers' Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon d
Inorganic and Structural Chemistry, Department of Chemistry, Bielefeld University, Bielefeld, Germany Published online: 04 Apr 2014.
To cite this article: Hugues Fouotsa, Simplice J.N. Tatsimo, Beate Neumann, Carmela Michalek, Celine Djama Mbazoa, Augustin Ephrem Nkengfack, Norbert Sewald & Alain Meli Lannang (2014) A new xanthone derivative from twigs of Garcinia nobilis, Natural Product Research: Formerly Natural Product Letters, 28:14, 1030-1036, DOI: 10.1080/14786419.2014.903398 To link to this article: http://dx.doi.org/10.1080/14786419.2014.903398
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Natural Product Research, 2014 Vol. 28, No. 14, 1030–1036, http://dx.doi.org/10.1080/14786419.2014.903398
A new xanthone derivative from twigs of Garcinia nobilis
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Hugues Fouotsaab, Simplice J.N. Tatsimoc, Beate Neumannd, Carmela Michalekb, Celine Djama Mbazoaa, Augustin Ephrem Nkengfacka, Norbert Sewaldb and Alain Meli Lannangbc* a Department of Organic Chemistry, Faculty of Science, University of Yaounde´ I, P.O. Box 812, Yaounde´, Cameroon; bDepartment of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany; cDepartment of Chemistry, Higher Teachers’ Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon; dInorganic and Structural Chemistry, Department of Chemistry, Bielefeld University, Bielefeld, Germany
(Received 20 January 2014; final version received 8 March 2014) Phytochemical investigation of the twigs of Garcinia nobilis led to the isolation of a new xanthone, named l-hydroxy-2,5-dimethoxyxanthone (1), together with 15 known compounds (2 – 16). The structures of the new and known compounds were established by means of spectroscopic methods and by comparison with previously reported data. The structure of compound 1 was confirmed by X-ray diffraction data. Compounds 1 –16 were tested for their cytotoxic activity against human cervix carcinoma cell line KB-3-1. Compounds 5 and 11 showed moderate activity while others showed weak biological activity in these cytotoxicity assays. Compounds 4 and 9 were found to be inactive. Keywords: Garcinia nobilis; Guttiferae; xanthone; cytotoxicity
1. Introduction Garcinia, one of the biggest genera of the family Guttiferae, has been found to be a rich source of xanthones (Bennett & Lee 1989), biflavonoids, benzophenones (Waterman & Hussain 1983) as well as triterpenoids (Nguyen & Harrison 2000). Phenolic constituents from Garcinia species have been reported to possess various biological activities, including antibacterial (Permana et al. 2001; Suksamrarn et al. 2003; Hay et al. 2004), cytotoxic (Shadid et al. 2007; Yu et al. 2009) and prooxidant (Wu et al. 2008) activities. They also displayed inhibitory activity against a-glucosidase, glycation (Fouotsa et al. 2012) and HIV (Gustafson et al. 1992). In line of our search for new and/or active substances from medicinal plants, we report here the chemical constituents of Garcinia nobilis, an endemic plant growing in the Zamangoue Forest, central region of Cameroon.
2. Results and discussion The methanol extract of twigs of G. nobilis was partitioned with petroleum ether and ethyl acetate (EtOAc). The petroleum ether extract was subjected to successive flash and column chromatography over silica gel and Sephadex to obtain a new xanthone derivative named l-hydroxy2,5-dimethoxyxanthone (1) and 15 other known compounds (2–16) identified as smeathxanthone A (2), 1,5-dihydroxy-3-methoxyxanthone (3), 1-hydroxy-3,6,7-trimethoxyxanthone (4),
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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1,3-dihydroxy-6,7-dimethoxyxanthone (5), 1,5-dihydroxyxanthone (6), euxanthone (7), 1hydroxy-7-methoxyxanthone (8), 1-hydroxy-5-methoxyxanthone (9), 1,3,7-trihydroxyxanthone (10), 1,3,5-trihydroxyxanthone (11), 1,3,6,7-tetrahydroxyxanthone (12), 1,3,5,6-tetrahydroxyxanthone (13), 30 ,6-dihydroxy-2,4,40 -trimethoxybenzophenone (14), friedelin (15) and 3ahydroxyfriedel-2-one (16) (Figure 1). Compound 1 was isolated as a yellow crystal. Its molecular formula C15H12O5 was deduced from HR-EI-MS. A positive test with alcoholic ferric chloride revealed its phenolic nature. The UV spectrum of this compound showed absorptions at lmax 240, 271, 303 and 313 nm indicating a trioxygenated xanthone (Ikeya et al. 1991). While the IR spectrum showed absorption bands at 1645 and 1580 cm21 corresponding to carbonyl group and aromatic ring (Ikeya et al. 1991). The 1H NMR spectrum of this compound 1 revealed the presence of two methoxy groups at dH 3.91 (s, 3H, 2-OMe) and 4,05 (s, 3H, 5-OMe). An AB system was observed with signals of aromatic protons at dH 7.54 (d, J ¼ 9.0 Hz, 1H, H-3) and 7.06 (d, J ¼ 9.0 Hz, 1H, H-4). Further analyses also indicated an aromatic ABC system with protons at dH 7.39 (t, J ¼ 8.1 Hz, 1H, H-7), 7.51 (brd, J ¼ 8.1 Hz, 1H, H-6) and 7.78 (dd, J ¼ 8.1; 1.3 Hz, 1H, H-8), respectively, suggesting the presence of three adjacent protons in the same ring. Further examination of 1H and 13C NMR spectra indicated signals at dH 12.77 (s, 1-OH) and dC 183.9 (C-9), suggesting the presence of a chelated hydroxyl and carbonyl group in 1. In the HMBC spectra of 1, the signal at dH 12.77 (1-OH) correlated with signals at dC 109.7 (C-9a), 143.9 (C-2) and 152.0 (C-1), while the methoxy groups at dH 3.91 (2-OMe) and 4.05 (5-OMe) showed long range correlation with carbons at dC 143.9 and 149.8, respectively, supporting
O
R7 R6
8
8a
R1 9a
B
R5
5
1
R2 2
A 10a O 4a
3 R 3
R4 1: R1 = OH; R2 = R4 = OMe; R3 = R5 = R6 = R7 = H 2: R1 = R3 = R4 = R7 = OH; R2 = geranyl; R5 = R6 = H 3: R1 = R4 = OH; R2 = R5 = R6 = R7 = H; R3 = OMe 4: R1 = OH; R2 = R4 = R7 = H; R3 = R5 = R6 = OMe 5: R1 = R3 = OH; R2 = R4 = R7 = H; R5 = R6 =OMe 6: R1 = R6 = OH; R2 = R3 = R4 = R5 = R7 = H 7: R1 = R4 = OH; R2 = R3 = R5 = R6 = R7 = H 8: R1 = OH; R2 = R3 = R5 = R6 = R7 = H; R4 = OMe 9: R1 = OH; R2 = R3 = R4 = R5 = R7 = H; R6 = OMe 10: R1 = R3 = R6 = OH; R2 = R4 = R5 = R7 = H 11: R1 = R3 = R4 = OH; R2 = R5 = R6 = R7 = H 12: R1 = R3 = R5 = R6 = OH; R2 = R4 = R7 = H 13: R1 = R3 = R4 = R5 = OH; R3 = R6 = R7 = H
O
OH
O O
O
O HO
O OH
14
Figure 1. Structure of isolated compounds.
15
16
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that ring A possesses a hydroxyl and a methoxy group at positions 1 and 2, respectively. This was further confirmed with the long range correlations observed between the proton at dH 7.54 (H-3) and carbons at dC 143.9 (C-2), 150.8 (C-4a) and 152.0 (C-1); the proton at dH 7.06 and carbons at dC 109.9 (C-9a), 143.9 (C-2) and 150.8 (C-4a). Finally, the correlations between dH 7.78 (H-8) and dC 117.6 (C-6), 147.8 (C-10a) and 183.9 (C-9) unambiguously indicated that B ring is substituted at position 5. Therefore, the structure of 1 (Figure 1), based on the aforementioned evidence, was elucidated as 1-hydroxy-2,5-dimethoxyxanthone. This structure was confirmed by X-ray analysis (Figure 2) The biological activity of compounds 1 – 16 was determined in cell-based cytotoxicity assays using the human cervix carcinoma cell line KB-3-1 in a resazurin assay (Sammet et al. 2010). Compounds 5 and 11 exhibited moderate IC50 values of 4.06 and 1.65 mM against KB-3-1. Other tested compounds showed weak activity while compounds 4 and 9 were inactive (Table 1). 3. Experimental 3.1. General IR spectra were recorded on a JASCO A-302 IR spectrophotometer (JASCO Labor-und Datentechnik GmbH, Gross-Umstadt, Germany). The 1H, 13C and 2D NMR spectra were recorded on a Bruker AMX-500 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) using CDCl3 as solvent. Homonuclear 1H – 1H connectivities were determined by using the COSY 458 experiment. One-bond 1H – 13C connectivities were determined by HMQC. Two and three-bond 1H – 13C connectivities were determined by HMBC experiments. Proton chemical shifts are reported in d (ppm) with reference to the residual CDCl3 signal at d 7.26, and 13C NMR spectra are referenced to the central peak of CDCl3 at d 77.0. Coupling constants (J) were measured in Hz. The EI-MS were recorded on a double-focusing mass spectrometer (Varian MAT 311A). HR-ESI-MS were recorded on a JEOL HX 110 mass spectrometer. Column chromatography was carried out on silica gel 60 (70 –230 and 240 – 300 mesh sizes, E. Merck, KGaA, Darmstadt, Germany), and with Sephadex LH-20 (GE Healthcare Europe GmbH, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Preparative thin layer chromatography (PTLC) was done on PTLC plates (E. Merck, F254). Precoated silica gel TLC was used to check the purity of compounds, and ceric sulphate spray reagent was used for visualisation of compounds by TLC.
Figure 2. The ORTEP plot of 1.
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Table 1. Cytotoxicity data of isolated compounds (1– 16).
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Compound
Concentration (mol/L)
IC50 (mol/L)
Comment
0.05 0.1 0.1 0.05 0.1 0.1 0.05 0.1 0.05 0.1 0.1 0.1 0.1 0.1 0.025 0.025
. 3.125 £ 1025 . 1.530 £ 1025 .1.25 £ 1024 NA 4.06 £ 1026 .6.25 £ 1025 .1.56 £ 1025 .6.25 £ 1024 NA .1.53 £ 1025 1.65 £ 1026 . 3.125 £ 1025 .5.9 £ 1024 .2.5 £ 1024 NA NA
A A A NA A A A A NA A A A A A NS NS
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Note: Range: from 1.25 £ 1024 to 7.629 £ 1029; A: active; NA: not active; NS: not soluble.
3.2. Plant material G. nobilis Engl. was collected in Okola, Central Province, Cameroon in April 2010, and identified by Mr Victor Nana of the Cameroon National Herbarium (Yaounde´) where a voucher specimen (50779/HNC/Cam/Mt Zamangoue´) has been deposited.
3.3. Extraction and isolation The air-dried and ground twigs of G. nobilis (1.5 kg) were extracted three times with MeOH (6.5 L) at room temperature. The resulting extract was concentrated under reduced pressure to obtain a crude extract (60.0 g). The obtained extract was partitioned with petroleum ether (2.5 L, 17.0 g) and EtOAc (2.5 L, 23.0 g). The petroleum ether extract (17.0 g) was then subjected to silica gel flash chromatography (8 cm £ 14 cm), eluting with petroleum ether – EtOAc of increasing polarity (10:0 1 L, 8:2 1 L, 6:4 750 mL, 4:6 500 mL, 0:10 500 mL). Fractions were subsequently combined based on their TLC profile into four fractions A –D. Fraction A (4.0 g) was subjected to silica gel (25 – 40 mm, 4.0 cm £ 60 cm) column chromatography eluting with petroleum ether – EtOAc with increasing polarity. Fractions of 25 mL were collected and subsequently combined on the basis of TLC profile into four fractions A1 – A4. Smeathxanthone A (2, 9 mg) and 1,5-dihydroxy-3-methoxyxanthone (3, 5 mg) were obtained from fraction A2 by using PTLC followed by further purification on silica gel chromatography (25 –40 mm; 3.0 cm £ 15 cm) eluting with petroleum ether –CH2Cl2 solvent system by increasing polarity. Fraction B (3.5 g) was further purified using column chromatography and silica gel (25 – 40 mm; 4.0 cm £ 60 cm) eluting with petroleum ether – EtOAc to obtain 1,3-dihydroxy-6,7-dimethoxyxanthone (5, 6 mg), friedelin (15) and 3ahydroxyfriedel-2-one (16). Fraction C (15 g) was subjected to column chromatography on silica gel (25 – 40 mm; 5 cm £ 20 cm) eluting with petroleum ether – EtOAc (150 mL), and the eluates were subsequently combined on the basis of the TLC profile to give four fractions C1 – C4. Fraction C2 (2 g) was further purified on silica gel column chromatography using petroleum ether –CH2Cl2 – MeOH by increasing polarity to afford 1-hydroxy-3,6,7-trimethoxyxanthone (4, 7 mg), 1,5-dihydroxyxanthone (6, 10 mg), euxanthone (7, 3 mg), 1-hydroxy-7-methoxyxanthone (8, 5 mg), 1-hydroxy-5-methoxyxanthone (9, 6 mg) and 1,3,7-trihydroxyxanthone
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(10, 5 mg). Fractions C3 and C4 (200 mg), similar on TLC, were combined and then subjected to silica gel column chromatography (25 – 40 mm; 3.0 £ 15 cm) using petroleum ether – CH2Cl2 – MeOH with increasing polarity to obtain the new xanthone (1, 3 mg) and 30 ,6dihydroxy-2,4,40 -trimethoxybenzophenone (14, 10 mg). Fraction D (4.0 g) was subjected to Sephadex LH-20 chromatography eluting with methanol. The resultant fraction was then subjected to PTLC and further purification was done using silica gel column chromatography (25 – 40 mm; 4.0 £ 60 cm) to obtain 1,3,5-trihydroxyxanthone (11, 4 mg), 1,3,6,7-tetrahydroxyxanthone (12, 3 mg) and 1,3,5,6-tetrahydroxyxanthone (13, 8 mg). 1-Hydroxy-2,5-dimethoxyxanthone (1): Yellow crystal; mp 175 – 1768C; UV (MeOH) lmax 240, 271, 303, 313 nm; IR (solid) nmax: 2921, 2851, 1733, 1645, 1619, 1580, 1494, 1456, 1438, 1270, 1232, 1191, 1155, 1074, 991, 772, 733, 676, 615 cm21; 1H NMR (500 MHz, CDCl3) d: 7.54 (d, J ¼ 9 Hz, 1H, H-3), 7.06 (d, J ¼ 9 Hz, 1H, H-4), 7.51 (br d, J ¼ 8.1 Hz, 1H, H-6), 7.39 (t, J ¼ 8.1 Hz, 1H, H-7), 7.78 (dd, J ¼ 8.1; 1.3 Hz, 1H, H-8), 3.91 (s, 3H, 2-OMe), 4.05 (s, 3H, 5-OMe), 12.77 (s, 1H, 1-OH) and 13C NMR (125 MHz, CDCl3) d: 152.0 (C-1), 143.9 (C-2), 123.9 (C-3), 106.8 (C-4), 149.8 (C-5), 117.6 (C-6), 124.7 (C-7), 117.0 (C-8), 183.9 (C-9), 150.8 (C-4a), 121.5 (C-8a), 109.9 (C-9a), 147.8 (C-10a), 57.9 (2-OMe), 56.9 (5-OMe); HR-ESI-MS m/ z: 567.12561 [2M þ Na]þ (calcd. for [(C15H12O5)2 þ Na]þ, 567.12677). Smeathxanthone A (2): Yellow powder; mp 216 –2188C; UV (EtOH) lmax: 408, 338, 337, 297, 223, 205 nm; IR (KBr) nmax: 3315, 2891, 1962, 1869, 1579, 1440, 1290, 1193 cm21 (Komguem et al. 2005). 1,5-Dihydroxy-3-methoxyxanthone (3): Yellow powder; mp: 269– 2718C; UV (NaOH) lmax: 240, 263, 278, 344 nm; IR (KBr) nmax: 3350, 1650, 1600, 1575 cm21 (Hashida et al. 2007). 1-Hydroxy-3,6,7-trimethoxyxanthone (4): Yellow powder; mp: 214 – 2158C; UV (MeOH) lmax: 241, 265, 314 nm; IR (KBr) nmax: 3372, 1648 cm21 (Ikeya et al. 1991). 1,3-Dihydroxy-6,7-dimethoxyxanthone (5): Yellow powder; mp 286 –2878C; UV (MeOH) lmax: 244, 310, 360 nm; IR (KBr) nmax: 3400, 1750 – 1650 cm21 (Liang et al. 2007). 1,5-Dihydroxyxanthone (6): Light yellow powder; mp 213– 2148C; UV (MeOH) lmax: 242, 363 nm; IR (KBr) nmax: 3275, 1645 cm21 (Kuete et al. 2007). Euxanthone (7): Yellow needles; mp 236– 2388C; UV (MeOH) lmax: 240, 360 nm; IR (KBr) nmax: 3250, 1640 cm21 (Trong et al. 2012). 1-Hydroxy-7-methoxyxanthone (8): Light yellow needles; mp 124– 1268C; UV (MeOH) lmax: 233, 259, 286, 340, 380 nm; IR (KBr) nmax: 3300, 1640 cm21 (Gunatilaka et al. 1982). 1-Hydroxy-5-methoxyxanthone (9): Light yellow needles; mp 217– 219; UV (MeOH) lmax: 251 (41.4), 311, 375 nm; IR (KBr) nmax: 3255, 1655, 1630, 1615, 1590, 1095 cm21 (Locksley & Murray 1971). 1,3,7-Trihydroxyxanthone (10): Light yellow powder, mp 215– 216; UV (MeOH) lmax: 238, 263, 323, 388 nm; IR (KBr) nmax: 3380, 1700, 1630 cm21 (Trong et al. 2012). 1,3,5-Trihydroxyxanthone (11): Light yellow needle; mp 302 –303; UV (NaOH) lmax: 257, 291, 348 nm; IR (KBr) nmax: 3375, 1655, 1612, 1580 cm21 (Locksley & Murray 1971). 1,3,6,7-Tetrahydroxyxanthone (12): Light yellow needle, mp 2708C; UV (MeOH) lmax: 207, 226, 299, 338 nm; IR (KBr) nmax: 3390, 1655 cm21 (Shibnath et al. 1973). 1,3,5,6-Tetrahydroxyxanthone (13): Yellow powder; mp 206 –207; UV (MeOH) lmax: 246, 282, 328 nm; IR (KBr) nmax: 3440, 1641, 1599 cm21 (Guat-Lee et al. 1995). 3 0 ,6-Dihydroxy-2,4,4 0 -trimethoxybenzophenone (14): Pale yellow crystal; mp 161 –1628C; UV (MeOH) lmax: 228, 280, 312 nm; IR (KBr) nmax: 3340, 1621, 1581, 1280, 1206, 1158 cm21 (Nguyen et al. 2005). Friedelin (15): White crystalline solid; mp 258 – 2608C; IR (KBr) lmax: 2927, 2870, 1715, 1463, 1390 cm21 (Li et al. 2012). 3a-Hydroxyfriedel-2-one (16): White crystalline solid; mp 233 –2348C; IR (KBr) lmax: 3460, 2960, 2880, 1460, 1390 cm21 (Qu et al. 2005).
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3.4. X-ray crystallographic study of 1 ˚ , b ¼ 15.1770(6) A ˚, Crystal data for 1. C15H12O5, M ¼ 272.25, monoclinic, a ¼ 4.8429(2) A 3 ˚ ˚ c ¼ 16.5317(9) A, b ¼ 93.568(5)8, V ¼ 1212.74(10) A , T ¼ 100 K, space group P21/n (no. 14), Z ¼ 4, m(Cu Ka) ¼ 0.949, 4557 reflections measured, 2130 unique (Rint ¼ 0.0263), which were used in all calculations. The final wR2 was 0.1325 (all data) and R1 was 0.0449 (I . 2ns(I)). CCDC 972523 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: þ 44 1223 336033; e-mail: [email protected]). 3.5. Cytotoxicity assays Cytotoxic activity screening of the isolates was done as described in previous reports (Sammet et al. 2010). The KB-3-1 cells were cultivated as a monolayer in Dulbecco’s modified Eagle medium with glucose (4.5 g/L), L -glutamine, sodium pyruvate and phenol red, supplemented with 10% (KB-3-1) foetal bovine serum (FBS). The cells were maintained at 378C in 5.3% CO2/humidified air. On the day before the test, the cells (70% confluence) were detached with trypsin solution (0.5%) and placed in sterile 96-well plates in a density of 10,000 cells in 100 mL medium per well. The dilution series of the compounds were prepared from stock solutions in DMSO of concentrations of 100 mM, 50 mM or 25 mM. The stock solutions were diluted with culture medium (10% FBS [KB-3-1]) down to pM range. The dilution prepared from stock solution was added to the wells. Each concentration was tested in six replicates. Dilution series were prepared by pipetting liquid from well to well. The control contained the same concentration of DMSO as the first dilution. After incubation for 72 h at 378C in 5.3% CO2/humidified air, 30 mL of an aqueous resazurin solution (175 mM) was added to each well. The cells were incubated under the same conditions for 5 h. Subsequently, the fluorescence (lex ¼ 530 nm) was measured at lem ¼ 588 nm. The IC50 values (the drug concentrations at which the cell viability is 50%) were calculated as a sigmoidal dose response curve using GRAPH PAD PRISM 4.03. 4. Conclusion The present work indicated that Garcinia nobilis is a good source of bioactive xanthones. Their cytotoxicity provided baseline information for the possible use of compounds 5 and 11 for the control of cancer diseases. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S5. Acknowledgements AML would like to thank the Alexander von Humboldt-Stiftung for support through Georg Forster Fellowship for Experienced Researchers (ID No. 1137675) at the University of Bielefeld (Germany). HF would like to acknowledge the German Academic Exchange Service (DAAD, Referat 412; PKZ A/12/904859) for a fellowship at the same University. This paper is also dedicated to the memory of Professor David Lontsi who passed away on 22 December 2008.
References Bennett GJ, Lee H. 1989. Xanthones from the Guttiferae. Phytochemistry. 28:967–998. Fouotsa H, Lannang AM, Mbazoa CD, Rasheed S, Bishnu PM, Zulfiqar A, Krishna PD, NKengfack AE, Farzana S, Muhammad IC, Sewald N. 2012. Xanthones inhibitors of a-glucosidase and glycation from Garcinia nobilis. Phytochem Lett. 5:236–239.
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Guat-lee S, Graham JB, Leslie JH, Keng-yeow S. 1995. Minor xanthones from the bark of Cratoxylum cochinchinense. Phytochemistry. 38:1521–1528. Gunatilaka AAL, Jasmin de silva AMY, Sotheeswara S. 1982. Minor xanthones of Hypericum mysorense. Phytochemistry. 21:1751–1753. Gustafson KR, Blunt JW, Munro MHG, Fuller RW, McKee TC, Cardellina JH, II, McMahon JB, Cragg GM, Boyd MR. 1992. The Guttiferones, HIV-inhibitory benzophenones from Symphonia globulifera, Garcinia livingstonei, Garcinia ovalifolia and Clusia rosea. Tetrahedron. 48:10093–10102. Hashida W, Tanaka N, Takaishi Y. 2007. Prenylated xanthones from Hypericum ascyron. J Nat Med. 61:371–374. Hay AE, Helesbeux JJH, Duval O, Labaı¨ed M, Grellier P, Richomme P. 2004. Antimalarial xanthones from Calophyllum caledonicum and Garcinia vieillardii. Life Sci. 75:3077–3085. Ikeya Y, Sugama K, Okada M, Mitsuhashi H. 1991. Two xanthones from Polygala tenuifolia. Phytochemistry. 30:2061–2065. Komguem J, Meli AL, Manfouo RN, Lontsi D, Ngounou FN, Kuete V, Kamdem HW, Tane P, Ngadjui BT, Sondengam BL, Connolly JD. 2005. Xanthones from Garcinia smeathmannii (Oliver) and their antimicrobial activity. Phytochemistry. 66:1713–1717. Kuete V, Meli AL, Komguem J, Louh GN, Tangmouo JG, Lontsi D, Meyer JJM, Lall N. 2007. Antimycobacterial, antibacterial and antifungal activities of the methanolic extract and compounds from Garcinia polyantha. Pharmacology Online. 3:87–95. Li F-F, Guo Z-Q, Chai X-Y, Tu P-F. 2012. Triterpenoids from the stems of Casearia velutina Bl. J Chin Pharm Sci. :273–277. Liang B, Li H-R, Xu L-Z, Yang S-L. 2007. Xanthones from the roots of Cudrania fruticosa Wight. J Asian Nat Prod Res. 9:393–397. Locksley HD, Murray LG. 1971. Extractives from Guttiferae. Part X1X. The isolation and structure of two benzophenones, six xanthones and two biflavonoids from the heartwood of Allanblackia floribunda Oliver. J Chem Soc. (C):1332–1340. Nguyen DLH, Harrison LJ. 2000. Xanthones and triterpenoids from the bark of Garcinia vilersiana. Phytochemistry. 53:111–114. Nguyen L-HD, Venkatraman G, Sim K-Y, Harrison LJ. 2005. Xanthones and benzophenones from Garcinia griffithii and Garcinia mangostana Nilar. Phytochemistry. 66:1718–1723. Permana D, Lajis NH, Mackeen MM, Ali AM, Aimi N, Kitajima M, Takayama H. 2001. Isolation and bioactivities of constituents of the roots of Garcinia atroviridis. J Nat Prod. 64:976– 979. Qu L, Chen X, Lu J, Yuan J, Zhao Y. 2005. Chemical components of leptopus chinensis. Chem Nat Compd. 41:565–568. Sammet B, Bogner T, Nahrwold M, Weiss C, Sewald N. 2010. Approaches for the Synthesis of functionalized cryptophycins. J Org Chem. 75:6953–6960. Shadid KA, Shaari K, Abas F, Israf DA, Hamzah AS, Syakroni N, Saha K, Lajis NH. 2007. Cytotoxic cagedpolyprenylated xanthonoids and a xanthone from Garcinia cantleyana. Phytochemistry. 68:2537–2544. Shibnath G, Ratan K, Chaudhuri. 1973. New tetra oxygenated xanthones of Canscora decussate. Phytochemtry. 12:2035–2038. Suksamrarn S, Suwannapoch N, Phakhodee W, Thanuhiranlert J, Ratananukul P, Chimnoi N, Suksamrarn A. 2003. Antimycobacterial activity of prenylated xanthones from the fruits of Garcinia mangostana. Chem Pharm Bull. 51:857–859. Trong TD, Thai TD, Phi HN, Eunhee K, Phuong TT, Won KO. 2012. Xanthones from Polygala karensium inhibit neuraminidases from influenza A viruses. Bioorg Med Chem Lett. 22:3688–3692. Waterman PG, Hussain RA. 1983. Systematic significance of xanthones, benzophenones and biflavonoids in Garcinia. Biochem Syst Ecol. 11:21–28. Wu CC, Lu YH, Wei BL, Yang SC, Won SJ, Lin CN. 2008. Phloroglucinols with prooxidant activity from Garcinia subelliptica. J Nat Prod. 71:246–250. Yu L, Zhao M, Yang B, Bai W. 2009. Immunomodulatory and anticancer activities of phenolics from Garcinia mangostana fruit pericarp. Food Chem. 116:969–973.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new xanthone derivative from twigs of Garcinia nobilis ab
c
d
Hugues Fouotsa , Simplice J.N. Tatsimo , Beate Neumann , b
a
Carmela Michalek , Celine Djama Mbazoa , Augustin Ephrem a
b
bc
Nkengfack , Norbert Sewald & Alain Meli Lannang a
Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon b
Department of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany c
Department of Chemistry, Higher Teachers' Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon d
Inorganic and Structural Chemistry, Department of Chemistry, Bielefeld University, Bielefeld, Germany Published online: 04 Apr 2014.
To cite this article: Hugues Fouotsa, Simplice J.N. Tatsimo, Beate Neumann, Carmela Michalek, Celine Djama Mbazoa, Augustin Ephrem Nkengfack, Norbert Sewald & Alain Meli Lannang (2014) A new xanthone derivative from twigs of Garcinia nobilis, Natural Product Research: Formerly Natural Product Letters, 28:14, 1030-1036, DOI: 10.1080/14786419.2014.903398 To link to this article: http://dx.doi.org/10.1080/14786419.2014.903398
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Natural Product Research, 2014 Vol. 28, No. 14, 1030–1036, http://dx.doi.org/10.1080/14786419.2014.903398
A new xanthone derivative from twigs of Garcinia nobilis
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Hugues Fouotsaab, Simplice J.N. Tatsimoc, Beate Neumannd, Carmela Michalekb, Celine Djama Mbazoaa, Augustin Ephrem Nkengfacka, Norbert Sewaldb and Alain Meli Lannangbc* a Department of Organic Chemistry, Faculty of Science, University of Yaounde´ I, P.O. Box 812, Yaounde´, Cameroon; bDepartment of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany; cDepartment of Chemistry, Higher Teachers’ Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon; dInorganic and Structural Chemistry, Department of Chemistry, Bielefeld University, Bielefeld, Germany
(Received 20 January 2014; final version received 8 March 2014) Phytochemical investigation of the twigs of Garcinia nobilis led to the isolation of a new xanthone, named l-hydroxy-2,5-dimethoxyxanthone (1), together with 15 known compounds (2 – 16). The structures of the new and known compounds were established by means of spectroscopic methods and by comparison with previously reported data. The structure of compound 1 was confirmed by X-ray diffraction data. Compounds 1 –16 were tested for their cytotoxic activity against human cervix carcinoma cell line KB-3-1. Compounds 5 and 11 showed moderate activity while others showed weak biological activity in these cytotoxicity assays. Compounds 4 and 9 were found to be inactive. Keywords: Garcinia nobilis; Guttiferae; xanthone; cytotoxicity
1. Introduction Garcinia, one of the biggest genera of the family Guttiferae, has been found to be a rich source of xanthones (Bennett & Lee 1989), biflavonoids, benzophenones (Waterman & Hussain 1983) as well as triterpenoids (Nguyen & Harrison 2000). Phenolic constituents from Garcinia species have been reported to possess various biological activities, including antibacterial (Permana et al. 2001; Suksamrarn et al. 2003; Hay et al. 2004), cytotoxic (Shadid et al. 2007; Yu et al. 2009) and prooxidant (Wu et al. 2008) activities. They also displayed inhibitory activity against a-glucosidase, glycation (Fouotsa et al. 2012) and HIV (Gustafson et al. 1992). In line of our search for new and/or active substances from medicinal plants, we report here the chemical constituents of Garcinia nobilis, an endemic plant growing in the Zamangoue Forest, central region of Cameroon.
2. Results and discussion The methanol extract of twigs of G. nobilis was partitioned with petroleum ether and ethyl acetate (EtOAc). The petroleum ether extract was subjected to successive flash and column chromatography over silica gel and Sephadex to obtain a new xanthone derivative named l-hydroxy2,5-dimethoxyxanthone (1) and 15 other known compounds (2–16) identified as smeathxanthone A (2), 1,5-dihydroxy-3-methoxyxanthone (3), 1-hydroxy-3,6,7-trimethoxyxanthone (4),
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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1,3-dihydroxy-6,7-dimethoxyxanthone (5), 1,5-dihydroxyxanthone (6), euxanthone (7), 1hydroxy-7-methoxyxanthone (8), 1-hydroxy-5-methoxyxanthone (9), 1,3,7-trihydroxyxanthone (10), 1,3,5-trihydroxyxanthone (11), 1,3,6,7-tetrahydroxyxanthone (12), 1,3,5,6-tetrahydroxyxanthone (13), 30 ,6-dihydroxy-2,4,40 -trimethoxybenzophenone (14), friedelin (15) and 3ahydroxyfriedel-2-one (16) (Figure 1). Compound 1 was isolated as a yellow crystal. Its molecular formula C15H12O5 was deduced from HR-EI-MS. A positive test with alcoholic ferric chloride revealed its phenolic nature. The UV spectrum of this compound showed absorptions at lmax 240, 271, 303 and 313 nm indicating a trioxygenated xanthone (Ikeya et al. 1991). While the IR spectrum showed absorption bands at 1645 and 1580 cm21 corresponding to carbonyl group and aromatic ring (Ikeya et al. 1991). The 1H NMR spectrum of this compound 1 revealed the presence of two methoxy groups at dH 3.91 (s, 3H, 2-OMe) and 4,05 (s, 3H, 5-OMe). An AB system was observed with signals of aromatic protons at dH 7.54 (d, J ¼ 9.0 Hz, 1H, H-3) and 7.06 (d, J ¼ 9.0 Hz, 1H, H-4). Further analyses also indicated an aromatic ABC system with protons at dH 7.39 (t, J ¼ 8.1 Hz, 1H, H-7), 7.51 (brd, J ¼ 8.1 Hz, 1H, H-6) and 7.78 (dd, J ¼ 8.1; 1.3 Hz, 1H, H-8), respectively, suggesting the presence of three adjacent protons in the same ring. Further examination of 1H and 13C NMR spectra indicated signals at dH 12.77 (s, 1-OH) and dC 183.9 (C-9), suggesting the presence of a chelated hydroxyl and carbonyl group in 1. In the HMBC spectra of 1, the signal at dH 12.77 (1-OH) correlated with signals at dC 109.7 (C-9a), 143.9 (C-2) and 152.0 (C-1), while the methoxy groups at dH 3.91 (2-OMe) and 4.05 (5-OMe) showed long range correlation with carbons at dC 143.9 and 149.8, respectively, supporting
O
R7 R6
8
8a
R1 9a
B
R5
5
1
R2 2
A 10a O 4a
3 R 3
R4 1: R1 = OH; R2 = R4 = OMe; R3 = R5 = R6 = R7 = H 2: R1 = R3 = R4 = R7 = OH; R2 = geranyl; R5 = R6 = H 3: R1 = R4 = OH; R2 = R5 = R6 = R7 = H; R3 = OMe 4: R1 = OH; R2 = R4 = R7 = H; R3 = R5 = R6 = OMe 5: R1 = R3 = OH; R2 = R4 = R7 = H; R5 = R6 =OMe 6: R1 = R6 = OH; R2 = R3 = R4 = R5 = R7 = H 7: R1 = R4 = OH; R2 = R3 = R5 = R6 = R7 = H 8: R1 = OH; R2 = R3 = R5 = R6 = R7 = H; R4 = OMe 9: R1 = OH; R2 = R3 = R4 = R5 = R7 = H; R6 = OMe 10: R1 = R3 = R6 = OH; R2 = R4 = R5 = R7 = H 11: R1 = R3 = R4 = OH; R2 = R5 = R6 = R7 = H 12: R1 = R3 = R5 = R6 = OH; R2 = R4 = R7 = H 13: R1 = R3 = R4 = R5 = OH; R3 = R6 = R7 = H
O
OH
O O
O
O HO
O OH
14
Figure 1. Structure of isolated compounds.
15
16
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that ring A possesses a hydroxyl and a methoxy group at positions 1 and 2, respectively. This was further confirmed with the long range correlations observed between the proton at dH 7.54 (H-3) and carbons at dC 143.9 (C-2), 150.8 (C-4a) and 152.0 (C-1); the proton at dH 7.06 and carbons at dC 109.9 (C-9a), 143.9 (C-2) and 150.8 (C-4a). Finally, the correlations between dH 7.78 (H-8) and dC 117.6 (C-6), 147.8 (C-10a) and 183.9 (C-9) unambiguously indicated that B ring is substituted at position 5. Therefore, the structure of 1 (Figure 1), based on the aforementioned evidence, was elucidated as 1-hydroxy-2,5-dimethoxyxanthone. This structure was confirmed by X-ray analysis (Figure 2) The biological activity of compounds 1 – 16 was determined in cell-based cytotoxicity assays using the human cervix carcinoma cell line KB-3-1 in a resazurin assay (Sammet et al. 2010). Compounds 5 and 11 exhibited moderate IC50 values of 4.06 and 1.65 mM against KB-3-1. Other tested compounds showed weak activity while compounds 4 and 9 were inactive (Table 1). 3. Experimental 3.1. General IR spectra were recorded on a JASCO A-302 IR spectrophotometer (JASCO Labor-und Datentechnik GmbH, Gross-Umstadt, Germany). The 1H, 13C and 2D NMR spectra were recorded on a Bruker AMX-500 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) using CDCl3 as solvent. Homonuclear 1H – 1H connectivities were determined by using the COSY 458 experiment. One-bond 1H – 13C connectivities were determined by HMQC. Two and three-bond 1H – 13C connectivities were determined by HMBC experiments. Proton chemical shifts are reported in d (ppm) with reference to the residual CDCl3 signal at d 7.26, and 13C NMR spectra are referenced to the central peak of CDCl3 at d 77.0. Coupling constants (J) were measured in Hz. The EI-MS were recorded on a double-focusing mass spectrometer (Varian MAT 311A). HR-ESI-MS were recorded on a JEOL HX 110 mass spectrometer. Column chromatography was carried out on silica gel 60 (70 –230 and 240 – 300 mesh sizes, E. Merck, KGaA, Darmstadt, Germany), and with Sephadex LH-20 (GE Healthcare Europe GmbH, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Preparative thin layer chromatography (PTLC) was done on PTLC plates (E. Merck, F254). Precoated silica gel TLC was used to check the purity of compounds, and ceric sulphate spray reagent was used for visualisation of compounds by TLC.
Figure 2. The ORTEP plot of 1.
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Table 1. Cytotoxicity data of isolated compounds (1– 16).
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Compound
Concentration (mol/L)
IC50 (mol/L)
Comment
0.05 0.1 0.1 0.05 0.1 0.1 0.05 0.1 0.05 0.1 0.1 0.1 0.1 0.1 0.025 0.025
. 3.125 £ 1025 . 1.530 £ 1025 .1.25 £ 1024 NA 4.06 £ 1026 .6.25 £ 1025 .1.56 £ 1025 .6.25 £ 1024 NA .1.53 £ 1025 1.65 £ 1026 . 3.125 £ 1025 .5.9 £ 1024 .2.5 £ 1024 NA NA
A A A NA A A A A NA A A A A A NS NS
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Note: Range: from 1.25 £ 1024 to 7.629 £ 1029; A: active; NA: not active; NS: not soluble.
3.2. Plant material G. nobilis Engl. was collected in Okola, Central Province, Cameroon in April 2010, and identified by Mr Victor Nana of the Cameroon National Herbarium (Yaounde´) where a voucher specimen (50779/HNC/Cam/Mt Zamangoue´) has been deposited.
3.3. Extraction and isolation The air-dried and ground twigs of G. nobilis (1.5 kg) were extracted three times with MeOH (6.5 L) at room temperature. The resulting extract was concentrated under reduced pressure to obtain a crude extract (60.0 g). The obtained extract was partitioned with petroleum ether (2.5 L, 17.0 g) and EtOAc (2.5 L, 23.0 g). The petroleum ether extract (17.0 g) was then subjected to silica gel flash chromatography (8 cm £ 14 cm), eluting with petroleum ether – EtOAc of increasing polarity (10:0 1 L, 8:2 1 L, 6:4 750 mL, 4:6 500 mL, 0:10 500 mL). Fractions were subsequently combined based on their TLC profile into four fractions A –D. Fraction A (4.0 g) was subjected to silica gel (25 – 40 mm, 4.0 cm £ 60 cm) column chromatography eluting with petroleum ether – EtOAc with increasing polarity. Fractions of 25 mL were collected and subsequently combined on the basis of TLC profile into four fractions A1 – A4. Smeathxanthone A (2, 9 mg) and 1,5-dihydroxy-3-methoxyxanthone (3, 5 mg) were obtained from fraction A2 by using PTLC followed by further purification on silica gel chromatography (25 –40 mm; 3.0 cm £ 15 cm) eluting with petroleum ether –CH2Cl2 solvent system by increasing polarity. Fraction B (3.5 g) was further purified using column chromatography and silica gel (25 – 40 mm; 4.0 cm £ 60 cm) eluting with petroleum ether – EtOAc to obtain 1,3-dihydroxy-6,7-dimethoxyxanthone (5, 6 mg), friedelin (15) and 3ahydroxyfriedel-2-one (16). Fraction C (15 g) was subjected to column chromatography on silica gel (25 – 40 mm; 5 cm £ 20 cm) eluting with petroleum ether – EtOAc (150 mL), and the eluates were subsequently combined on the basis of the TLC profile to give four fractions C1 – C4. Fraction C2 (2 g) was further purified on silica gel column chromatography using petroleum ether –CH2Cl2 – MeOH by increasing polarity to afford 1-hydroxy-3,6,7-trimethoxyxanthone (4, 7 mg), 1,5-dihydroxyxanthone (6, 10 mg), euxanthone (7, 3 mg), 1-hydroxy-7-methoxyxanthone (8, 5 mg), 1-hydroxy-5-methoxyxanthone (9, 6 mg) and 1,3,7-trihydroxyxanthone
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(10, 5 mg). Fractions C3 and C4 (200 mg), similar on TLC, were combined and then subjected to silica gel column chromatography (25 – 40 mm; 3.0 £ 15 cm) using petroleum ether – CH2Cl2 – MeOH with increasing polarity to obtain the new xanthone (1, 3 mg) and 30 ,6dihydroxy-2,4,40 -trimethoxybenzophenone (14, 10 mg). Fraction D (4.0 g) was subjected to Sephadex LH-20 chromatography eluting with methanol. The resultant fraction was then subjected to PTLC and further purification was done using silica gel column chromatography (25 – 40 mm; 4.0 £ 60 cm) to obtain 1,3,5-trihydroxyxanthone (11, 4 mg), 1,3,6,7-tetrahydroxyxanthone (12, 3 mg) and 1,3,5,6-tetrahydroxyxanthone (13, 8 mg). 1-Hydroxy-2,5-dimethoxyxanthone (1): Yellow crystal; mp 175 – 1768C; UV (MeOH) lmax 240, 271, 303, 313 nm; IR (solid) nmax: 2921, 2851, 1733, 1645, 1619, 1580, 1494, 1456, 1438, 1270, 1232, 1191, 1155, 1074, 991, 772, 733, 676, 615 cm21; 1H NMR (500 MHz, CDCl3) d: 7.54 (d, J ¼ 9 Hz, 1H, H-3), 7.06 (d, J ¼ 9 Hz, 1H, H-4), 7.51 (br d, J ¼ 8.1 Hz, 1H, H-6), 7.39 (t, J ¼ 8.1 Hz, 1H, H-7), 7.78 (dd, J ¼ 8.1; 1.3 Hz, 1H, H-8), 3.91 (s, 3H, 2-OMe), 4.05 (s, 3H, 5-OMe), 12.77 (s, 1H, 1-OH) and 13C NMR (125 MHz, CDCl3) d: 152.0 (C-1), 143.9 (C-2), 123.9 (C-3), 106.8 (C-4), 149.8 (C-5), 117.6 (C-6), 124.7 (C-7), 117.0 (C-8), 183.9 (C-9), 150.8 (C-4a), 121.5 (C-8a), 109.9 (C-9a), 147.8 (C-10a), 57.9 (2-OMe), 56.9 (5-OMe); HR-ESI-MS m/ z: 567.12561 [2M þ Na]þ (calcd. for [(C15H12O5)2 þ Na]þ, 567.12677). Smeathxanthone A (2): Yellow powder; mp 216 –2188C; UV (EtOH) lmax: 408, 338, 337, 297, 223, 205 nm; IR (KBr) nmax: 3315, 2891, 1962, 1869, 1579, 1440, 1290, 1193 cm21 (Komguem et al. 2005). 1,5-Dihydroxy-3-methoxyxanthone (3): Yellow powder; mp: 269– 2718C; UV (NaOH) lmax: 240, 263, 278, 344 nm; IR (KBr) nmax: 3350, 1650, 1600, 1575 cm21 (Hashida et al. 2007). 1-Hydroxy-3,6,7-trimethoxyxanthone (4): Yellow powder; mp: 214 – 2158C; UV (MeOH) lmax: 241, 265, 314 nm; IR (KBr) nmax: 3372, 1648 cm21 (Ikeya et al. 1991). 1,3-Dihydroxy-6,7-dimethoxyxanthone (5): Yellow powder; mp 286 –2878C; UV (MeOH) lmax: 244, 310, 360 nm; IR (KBr) nmax: 3400, 1750 – 1650 cm21 (Liang et al. 2007). 1,5-Dihydroxyxanthone (6): Light yellow powder; mp 213– 2148C; UV (MeOH) lmax: 242, 363 nm; IR (KBr) nmax: 3275, 1645 cm21 (Kuete et al. 2007). Euxanthone (7): Yellow needles; mp 236– 2388C; UV (MeOH) lmax: 240, 360 nm; IR (KBr) nmax: 3250, 1640 cm21 (Trong et al. 2012). 1-Hydroxy-7-methoxyxanthone (8): Light yellow needles; mp 124– 1268C; UV (MeOH) lmax: 233, 259, 286, 340, 380 nm; IR (KBr) nmax: 3300, 1640 cm21 (Gunatilaka et al. 1982). 1-Hydroxy-5-methoxyxanthone (9): Light yellow needles; mp 217– 219; UV (MeOH) lmax: 251 (41.4), 311, 375 nm; IR (KBr) nmax: 3255, 1655, 1630, 1615, 1590, 1095 cm21 (Locksley & Murray 1971). 1,3,7-Trihydroxyxanthone (10): Light yellow powder, mp 215– 216; UV (MeOH) lmax: 238, 263, 323, 388 nm; IR (KBr) nmax: 3380, 1700, 1630 cm21 (Trong et al. 2012). 1,3,5-Trihydroxyxanthone (11): Light yellow needle; mp 302 –303; UV (NaOH) lmax: 257, 291, 348 nm; IR (KBr) nmax: 3375, 1655, 1612, 1580 cm21 (Locksley & Murray 1971). 1,3,6,7-Tetrahydroxyxanthone (12): Light yellow needle, mp 2708C; UV (MeOH) lmax: 207, 226, 299, 338 nm; IR (KBr) nmax: 3390, 1655 cm21 (Shibnath et al. 1973). 1,3,5,6-Tetrahydroxyxanthone (13): Yellow powder; mp 206 –207; UV (MeOH) lmax: 246, 282, 328 nm; IR (KBr) nmax: 3440, 1641, 1599 cm21 (Guat-Lee et al. 1995). 3 0 ,6-Dihydroxy-2,4,4 0 -trimethoxybenzophenone (14): Pale yellow crystal; mp 161 –1628C; UV (MeOH) lmax: 228, 280, 312 nm; IR (KBr) nmax: 3340, 1621, 1581, 1280, 1206, 1158 cm21 (Nguyen et al. 2005). Friedelin (15): White crystalline solid; mp 258 – 2608C; IR (KBr) lmax: 2927, 2870, 1715, 1463, 1390 cm21 (Li et al. 2012). 3a-Hydroxyfriedel-2-one (16): White crystalline solid; mp 233 –2348C; IR (KBr) lmax: 3460, 2960, 2880, 1460, 1390 cm21 (Qu et al. 2005).
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3.4. X-ray crystallographic study of 1 ˚ , b ¼ 15.1770(6) A ˚, Crystal data for 1. C15H12O5, M ¼ 272.25, monoclinic, a ¼ 4.8429(2) A 3 ˚ ˚ c ¼ 16.5317(9) A, b ¼ 93.568(5)8, V ¼ 1212.74(10) A , T ¼ 100 K, space group P21/n (no. 14), Z ¼ 4, m(Cu Ka) ¼ 0.949, 4557 reflections measured, 2130 unique (Rint ¼ 0.0263), which were used in all calculations. The final wR2 was 0.1325 (all data) and R1 was 0.0449 (I . 2ns(I)). CCDC 972523 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: þ 44 1223 336033; e-mail: [email protected]). 3.5. Cytotoxicity assays Cytotoxic activity screening of the isolates was done as described in previous reports (Sammet et al. 2010). The KB-3-1 cells were cultivated as a monolayer in Dulbecco’s modified Eagle medium with glucose (4.5 g/L), L -glutamine, sodium pyruvate and phenol red, supplemented with 10% (KB-3-1) foetal bovine serum (FBS). The cells were maintained at 378C in 5.3% CO2/humidified air. On the day before the test, the cells (70% confluence) were detached with trypsin solution (0.5%) and placed in sterile 96-well plates in a density of 10,000 cells in 100 mL medium per well. The dilution series of the compounds were prepared from stock solutions in DMSO of concentrations of 100 mM, 50 mM or 25 mM. The stock solutions were diluted with culture medium (10% FBS [KB-3-1]) down to pM range. The dilution prepared from stock solution was added to the wells. Each concentration was tested in six replicates. Dilution series were prepared by pipetting liquid from well to well. The control contained the same concentration of DMSO as the first dilution. After incubation for 72 h at 378C in 5.3% CO2/humidified air, 30 mL of an aqueous resazurin solution (175 mM) was added to each well. The cells were incubated under the same conditions for 5 h. Subsequently, the fluorescence (lex ¼ 530 nm) was measured at lem ¼ 588 nm. The IC50 values (the drug concentrations at which the cell viability is 50%) were calculated as a sigmoidal dose response curve using GRAPH PAD PRISM 4.03. 4. Conclusion The present work indicated that Garcinia nobilis is a good source of bioactive xanthones. Their cytotoxicity provided baseline information for the possible use of compounds 5 and 11 for the control of cancer diseases. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S5. Acknowledgements AML would like to thank the Alexander von Humboldt-Stiftung for support through Georg Forster Fellowship for Experienced Researchers (ID No. 1137675) at the University of Bielefeld (Germany). HF would like to acknowledge the German Academic Exchange Service (DAAD, Referat 412; PKZ A/12/904859) for a fellowship at the same University. This paper is also dedicated to the memory of Professor David Lontsi who passed away on 22 December 2008.
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